Proper muscle regeneration is necessary to restore normal muscle architecture and function after traumatic injury. Aberrant regeneration can result in alterations in myofiber size, number and architecture. Myofibers with an abnormal branching cytoarchitecture are commonly found in various neuromuscular diseases as well as after severe muscle injury in various species, including human. These aberrant myofibers are fragile and a correlation exists between muscles containing a high percentage of these myofibers and weakness and increased incidence of injury. Muscles containing high levels of branched myofibers are unlikely to function in a normal physiologic manner. To date the mechanisms and molecules regulating myofiber branching have been obscure. The mechanisms regulating myofiber branching are significant to elucidate from both a basic and a clinical standpoint. We recently established a novel role for mouse odorant receptor 23 (MOR23) in skeletal muscle regeneration in mice and identified the first molecule with a functional role in myofiber branching. Odorant receptor signaling in olfactory neurons regulates expression of several adhesion and chemoattractant molecules through an adenylyl cyclase 3 (AC3) and protein kinase A (PKA) dependent signaling pathway. The downstream molecules regulated by MOR23 signaling in skeletal muscle that play a role in myofiber branching are unknown. This proposal will elucidate the molecular pathway by which MOR23 regulates myofiber branching in mice. Based on our preliminary data we hypothesize that activation of MOR23 in skeletal muscle stimulates AC3 and protein kinase A (PKA) signaling leading to changes in downstream molecules that regulate muscle cell migration and adhesion and thus, myofiber branching. We will use various genetic mouse models to test the roles of these different molecules in regulating muscle cell migration and adhesion as well as myofiber branching. The proposed experiments are novel because they are the first to mechanistically dissect regulation of myofiber branching. Odorant receptor signaling pathways and their downstream molecular effectors may serve as effective pharmacologic targets for decreasing myofiber branching in various neuromuscular disorders. Decreasing the number of branched myofibers will likely be beneficial for improving both muscle physiology and the efficiency of cell and gene therapy approaches for muscular disorders. PUBLIC HEALTH RELEVANCE: Muscle cells with an abnormal branching shape are commonly found in various muscle diseases as well as after severe muscle injury. These abnormal muscle cells are fragile and muscles containing a high percentage of these types of cells are weaker and become injured more easily. How these branched muscle cells arise is unknown. The mechanisms regulating the formation of these abnormal muscle cells is important to define from both a basic science and a clinical standpoint. We will use biochemical and genetic methods to manipulate the function of several molecules we hypothesize to play a role in muscle cell branching and analyze the outcome. The proposed experiments are novel because they are the first to mechanistically dissect regulation of muscle cell branching. In the future, the pathway under study may lead to drug therapies for eliminating these abnormal muscle cells in various muscle disorders.